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Abstract

The encompassing aim of this project was to gain a better understanding of the role of the cellular immune response to dengue virus (DV) infection. Dengue virus occurs as four distinct serotypes, called D1-D4. Symptomatic DV infection occurs as two forms of illness. The more severe form of DV infection, dengue hemorrhagic fever (DHF), is characterized by increased capillary permeability resulting in decreased plasma volume, which may be accompanied by hemorrhagic manifestations. At its most severe, DHF can result in circulatory shock and death. Epidemiological studies indicate that DHF is more likely to occur following a secondary infection with a serotype of DV other than that which caused the primary infection, and there is evidence of increased T cell activation in more severe disease. These data and others indicate that DHF may be of an immunopathological nature.

The memory CD4+ T cell response of a D4-immune donor was analyzed. Bulk culture proliferative responses of peripheral blood mononuclear cells (PBMC) to noninfectious DV antigens showed the highest proliferation to D4V antigen, with lesser, crossreactive proliferation to D2V antigen. CD4+ cytotoxic T lymphocyte (CTL) clones were established by stimulation with D4 antigen using a limiting dilution method. Seven out of 15 clones recognized the D4V capsid protein. The clones showed heterogeneity in their usage of T cell receptor Vα and Vβ genes. Six of these CTL clones were crossreactive between 02 and 04, and one clone was specific for D4. Using synthetic peptides, the D4V-specific clone was found to recognize an epitope between amino acids (aa) 47-55 of the capsid protein, while the crossreactive CTL clones each recognized epitopes in a separate location, between aa 83 and 92, which is conserved between D2 and D4. These results showed that the DV capsid protein can be a target of the cellular immune response following DV infection.

The bulk culture response of the donor's PBMC to the epitope peptide spanning aa 84-92 was also examined. Peptides containing this epitope induced proliferation of the donor's PBMC in bulk culture, but peptides not containing the entire epitope did not induce proliferation. Also, PBMC stimulated in bulk culture with noninfectious D4V antigen lysed autologous target cells pulsed with peptides containing aa 84-92. These results indicate that this donor exhibits memory CD4+ T cell responses directed against the DV capsid protein and suggest that the response to the capsid protein is dominant not only in vitro at the clonal level, but in bulk culture responses as well.

Experiments were performed demonstrating that the CD4+ CTL clones were capable of mediating bystander lysis of non-antigen presenting target cells. Following activation on plate-bound anti-CD3 antibody or in the presence of unlabeled antigen-presenting target cells, these clones could lyse both Jurkat cells and HepG2 cells as bystander targets. Bystander lysis of neighboring, non-infected cells by activated CD4+ CTL clones might contribute to the pathology of DHF. The mechanisms of lysis employed by the T cell clones against both cognate and bystander target cells were assessed using chemical inhibitors of either the perforin- or Fas/FasL-mediated pathways. Three CD4+ CTL clones were demonstrated to lyse cognate, antigen-presenting target cells by a mechanism that primarily involves perforin, while bystander lysis occurred through Fas/FasL interactions. In contrast, one clone used a Fas/FasL mechanism to lyse both cognate and bystander targets. These experiments indicated that the perforin- and FasL-mediated mechanisms of target cell lysis are not mutually exclusive, in that a single clone can kill target cells using either mechanism. Additionally, the ability of CD4+ CTL clones to lyse target cells by the perforin pathway indicates that, like CD8+ CTL, these clones might play a role in viral clearance and recovery from infection through lysis of virus-infected cells.

Cytokine production by the capsid-specific CTL clones was also examined. Six of six clones studied produced high quantities of IFN-γ in response to either D2V antigen or the epitope peptide. IFN-γ was also produced by PBMC in a bulk culture from this donor stimulated with D4V antigen. All of the clones produced both TNF-α and TNF-β following stimulation. Four of six clones produced low amounts of IL-2, and only three of six clones produced detectable amounts of IL-4. Production of cytokines by activated CD4+ T cell clones in vivo could contribute to both viral clearance and immunopathology.

To better understand the role that cytokine production might play in vivo in response to DV infection, cytokine mRNA levels were examined by PCR in DV-infected Thai children. mRNA for the cytokines IFN-γ, TNF-β, TNF-α, IL-1β, and IL-6 were detectable in the PBMC of DV-infected children. Semi-quantitative PCR analysis indicated that TNF-α mRNA levels were elevated in Thai children with DHF compared to children with classical dengue fever, the less severe form of illness (p=.013). All other cytokines showed no statistically significant difference between children with DHF and those with DF, although IFN-γ showed a trend toward elevation in more severe disease (p=.l). Increased production of TNF-α and/or IFN-γ in vivo could potentially contribute to the immunopathology of severe dengue illness.

Taken as a whole, the data presented in this thesis provide a better understanding of the role of the cellular immune response to dengue virus infection and its potential contribution to the immunopathology of dengue hemorrhagic fever.